This R01 application, over its funded cycles, articulated and substantiated the thesis that the inducible heme- degrading enzyme, heme oxygenase-1 (HO-1), protects against AKI induced by ischemia-reperfusion (IR), heme proteins, and inflammation; this R01 also demonstrated that HO-1 and its products protect against hypertensive and vascular injury. Evidence is emerging that 1) intracellular and extracellular levels of heme, a nephrotoxin, are increased in AKI, thereby providing one of the final common pathways for AKI; and 2) the paradigm exemplified by HO-1 applies to other heme-related proteins: namely, HO-1 is the forerunner for a family of proteins that are not only of pathophysiologic significance beyond heme metabolism, but may provide new therapeutic strategies for AKI. This renewal focuses on 3 such heme-related proteins: hemopexin (HPX), the high-affinity, heme-binding protein; HO-2, the constitutive heme-degrading protein; and haptoglobin (Hp), the high-affinity, hemoglobin-binding protein. Aim #1 tests the hypothesis that HPX is an inducible, heme- binding protectant against AKI. The effects of HPX protein and genetic deficiency of HPX, singly and in combination, will be examined in: 1) cisplatin-induced and IR-induced AKI; 2) heme-induced apoptosis and inhibition of cell proliferation, and cisplatin-induced apoptosis; and 3) heme-induced inflammation, the latter involving TLR4 signaling. Aim #2 hypothesizes that constitutive HO-2 is an unrecognized protectant against AKI. Using HO-2-/- mice, this aim will examine whether the absence of the restraining effect of HO-2 exaggerates heme/NF-kB-dependent, inflammatory responses, and the extent to which TLR4 is involved. The role of HO-2 in suppressing heme-driven discharge of the inflammatory and vasoactive constituents of the Weibel-Palade Bodies (WPBs) will be examined. This aim will also determine whether HO-2 deficiency exacerbates vascular behavior in AKI, and whether a product of HO, namely CO, attenuates such effects. Aim #3 hypothesizes that haptoglobin (Hp) is an inducible protectant against AKI. Hp is now known to possess antioxidant effects, besides its binding of hemoglobin. Aim #3 will examine whether Hp-/- mice exhibit increased sensitivity to IR-induced AKI and the capacity of Hp protein to reduce such sensitivity, focusing on the antioxidant actions of Hp; the efficacy of Hp in interrupting the prooxidant effects of heme in vitro will also be testd. Finally, this aim will examine whether mutant Hp 2-2 mice, expressing a disease-prone human genotype, exhibit increased sensitivity to IR-induced AKI due to impaired antioxidant actions of Hp 2-2. In summary, this application explores 3 novel, renal protective pathways and elucidates the importance of: heme as a common pathway for AKI and the role of heme-binding as a protective strategy; constitutive heme- degrading mechanisms in protecting against AKI; heme, by activating TLR4, as a novel damage-associated molecular pattern (DAMP); heme as a agonist for release of WPBs in AKI; and Hp, a protein in use or considered for use in human disease, as a novel, broadly applicable, but as yet untested therapy for AKI.